MXPA01000386A - Pharmaceutical uses of nab1 and nab2 - Google Patents

Abstract

The invention relates to the use, particularly in gene therapy, of an NAB1 or NAB2 polypeptide or a biologically active fragment thereof, and to nucleic acid molecules encoding such polypeptides, in the manufacture of a medicament for the treatment of cell proliferation disorders associated with wound healing in a mammal, including human.

Description

- IL ¬ PHARMACEUTICAL USES OF NAB1 AND MAB2 The invention relates to the use of techniques in gene therapy in wound healing. More particularly, it refers to the new use of polynucleotides encoding NAB1 or NAB2 transcriptional repressor proteins in the down-regulation of cell proliferative conditions, particularly the slow healing of the skin to prevent the formation of hypertrophic and keloid scar, psoriasis, inhibition of restenosis followed by percutaneous trans-luminal coronary angioplasty, modulation of the calcification of the vessel wall and inhibition of cell proliferation in cancer.

Skin healing involves a wide range of cellular, molecular, physiological and biochemical events. During the healing process, the cells migrate to the wound sites where they proliferate and synthesize the components of the extracellular matrix to reconstitute a narrow tissue similar to the intact original. This activity is regulated by secretory mediators of cells at the edge of wounds such as platelet-derived platelet growth factor (PDGF), epidermal growth factor (EGF), growth factor beta Transformant (TGF beta) and other cytokines. Beneficial effects of these agents have been demonstrated both in vitro and in vivo cells (reviewed by Moulin, Eur. J. Cell Biol. 68, 1-7, 1995), which include the benefit of administering PDGF in rat models with diabetes (Brown et al J. Surg, Res. 56; 562-570, 1994).

It has been demonstrated in the past five years, that numerous growth factors accelerate cell proliferation in vi tro and promote wound healing in animal models. TGF beta has received great attention in the context of wound repair as it promotes cell proliferation, differentiation and matrix production. TGF beta administered either topically or systematically accelerates the speed of repair of skin wounds in animal models. (Ashcroft et al Nature Medicine, 3; 1209-1215, 1997; Sporn and Roberts J. Cell Biol. 119; 1017-1021, 1997; Beck et al., J. Clin., Invest. 92; 2841-2849, 1993). It has also been reported that PDGF promotes re-epithelialization and revascularization in ischemic tissue and in animals with diabetes (Uhl et al Langenbecks Archiv fur Chirurgie-Supplement-Krongressband 114; 705-708, 1997 and reviewed in Dirks and Bloemers Mol. Biol. Reports 22; 1-24, 1996).

- The Egr-1 transcription factor (early growth response gene) is a potent regulator of around 30 genes and plays a role in cell proliferation, development and differentiation (reviewed in Liu et al 5 Cpt. Rev. Oncogenesis 7; 101-125, 1996; Khachigian and Collins Circ Res. 81; 457-461, 1997). Egr-1 is induced after damage to the vascular endothelium (eg, Khachigian et al Science; 271, 1427-1431, 1996) and the targets for transcriptional activation are numerous genes that include EGF, factor A of platelet-derived growth (PDGF-A), basic fibroblast growth factor (bFGF), induction of PDGF A, platelet-derived growth factor-B (PDGF B), TGF beta, bFGF, uro-plasminogen activator (u-PA), tissue factor and factor-2 of growth similar to insulin (IGF-2). The blockade of Egr-1 inducible in TGF beta can have application in the prevention of scar formation. Antibodies raised against TGF beta reduce wound healing in incisional wounds in rodents (Shah et al J. Cell Science; 107; 1137-1157; Shah et al Lancet; 339, 213-214, 1992).

The transcription complex mediating the induction of vascular endothelial growth factor (VEGF) depends on AP2 and not directly on Egr-1 (Gille et al EMBO J 16; 750- 759, 1997). However, VEGF expression directly overregulates PDGF (Finkenzeller Oncogene 15; 669-676, 1997). Transcription of VEGF mRNA is enhanced by a number of factors including PDGF B, bFGF, keratinocyte growth factor (KGF), EGF, tumor necrosis factor (TNF) alpha and TGF beta 1. In animal models, have shown that VEGF-activated passivation of metal rods inhibits neo-intimal formation, accelerates reendothelization and elevates vasomotor activity (Asahara et al Circulation; 94, 3291-3302).

The VEGF expression vector has been reported in wound healing and psoriatic skin, both conditions in which TGF alpha and its ligand, EGFr are upregulated. Expression of EGF induces Egr-1 (Iwami et al Am. J. Physiol., 270; H2100-2107, 1996; Fang et al. Calcified Tissue International 57; 450-455, 1995; J. Neurosciences Res. 36; 58-65 , 1993). There is anecdotal evidence in the present that Egr-1 can activate the expression of the intercellular adhesion of molecule -1 (ICAM-1) in B lymphocytes stimulated by phorbol ester (Maltzman et al., Cell Biol; 16; 2283- 2294, 1996) and can activate the expression of TNF alpha by virtue of the presence of a site that binds Egr-1 in the TNF alpha promoter (Kramer et al. Biochim. Biophys.

A ^ ¿m? S ^ ..JGi & ^ "&« ñ! < ! íisb. i. ~ ^ .sr, '-, n? ! 4 ** v < . .feassa. - - - 1219; 413-421, 1994). Finally, mice blocked with Egr-1 are infertile and deficient in leuteinizing hormone (LH) (Lee et al 273; 1219-121, 1996) which ims that the LH promoter may also be an objective for the activation of Egr-1. Vascular calcification is an actively regulated process similar to bone formation that involves cells and factors known to be important in the regulation of bone metabolism (reviewed in Dermer et al Trends Cardiovascular, Med. 4; 45-49). , 1994). Regulators of osteoblastogenesis and / or osteoclastogenesis can modulate the degree of calcification of the vessel walls. The NAB, NAB1 and NAB2 proteins (corepressors that bind NGFI-A) interact with the conserved Rl domain of the Egr-1 and Egr-2 transactivators (Svaren et al EMBO J., 17; 6010-6019, 1998). It has previously been shown that NAB2 will repress the NGF-induced differentiation of PC12 cells (Qu, Z. et al J. Cell Biol. 142; 1075-1082, 1998) and the Egr-1-mediated activation of basic FGF (Svaren et al. to EMBO J., 17; 6010-6019, 1998).

A problem that is found in wound healing is the formation of hypertrophic and keloid scars. This is extremely undesirable, particularly after cosmetic surgery. Tissue fibrosis (for example * & ß * t j &¥ »? ft? s? t¡? ttS ¡) gí > t *. -., & »R * s * JBMU» '»á"? - - apd to the kidney, liver or skin) is manifested by the accumulation of extracellular matrix.The dysregulated production of the extracellular matrix that supports the development of tissue fibrosis is regulated at least in part, mainly by a number of growth factors, but not restricted to TGFβ (Muir Eur. J. Plast Surg. 21; 1-7, 1998, PDGF isomorphs (Katou et al J. Pathol.; 201-208, 1998, Heldin et al in The molecular and cellular biology of wound repair, Clark, ed.; 249-264, 1966 Plenum Press) and VEGF (Jones et al Frontiers in Bioscience 4; D303-309, 1999 A therapy that reduces it, but does not eliminate the expression of growth factors in the wound site, would have a significant impact on the healing of scar tissue reduced and improvement in quality of life.

The International Patent Apation number PCT. GB99.01722 describes the use of the Egr-1 transcription factor in which it promotes wound healing. The present inventors have now found that administration of a polynucleotide encoding the transcriptional repressor NAB1 or NAB2: (a) represses the mediated activation of Egr-1 of the growth factors in vi tro; (b) that represses the basal levels of expression of growth factor in m vi tro genes; and (c) at the site of wounds in a rodent, and the subsequent expression thereof, reduces the expression of TGF-β1 but not that of TGF-β3 and has an apation for the reduction of healing during healing (eg, Shah et al J. Cell Science 107; 1137-1157, 1994).

Therefore, the sub-regulation of Egr-1 decreases the healing process and reduces the incidence of hypertrophic and keloid scar formation and psoriasis. The sub-regulation of Egr-1 may also be useful in the treatment of other conditions associated with the healing of wounds, such as restenosis followed by percutaneous trans-luminal coronary angiosplasty, modulation in the calcification of the walls of the vessels. inhibition of cell proliferation in cancer.

Thus, according to one aspect of the invention, there is provided the use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a medicament for the treatment of cell proliferative diseases associated with the healing of wounds in a mammal, including human.

According to a further aspect of the invention, it provides a method for the treatment of cellular proliferative diseases associated with wound healing in a mammal, including human, which comprises administering to the mammal a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof.

According to a further aspect, the invention provides a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof for use in the treatment of cellular proliferative conditions associated with wound healing.

As a further aspect, the invention extends the use of a combination of a nucleic acid molecule comprising a sequence encoding a NAB1 polypeptide and another comprising a sequence encoding a NAB2 polypeptide.

As a preferred aspect of the present invention, the cell proliferative conditions associated with wound healing are selected from the formation of hypertrophic and keloid scar, psoriasis, restenosis followed by percutaneous trans-luminal coronary angioplasty, calcification in the walls of the vessels and cell proliferation in cancer. As a particularly preferred aspect of the present invention, cell proliferative diseases are, hypertrophic and keloid scar formation.

Thus, the present invention relates to the therapeutic use in wound healing processes of polynucleotides encoding NAB1 or NAB2. The invention also relates to therapeutic use in wound healing processes of NAB1 or NAB2 itself, as described in more detail below.

The invention relates to the use of NAB1 or NAB2 polypeptides and nucleic acid sequences encoding NAB1 or NAB2 polypeptides of any origin or species. The human DNA sequence is listed in Genbank under accession number U47007, which encodes a nuclear protein of 486 amino acids. The NAB1 rat is a nuclear protein of 570 amino acids and is described in PNAS USA 92, 1995 p6873-6877. The rat DNA sequence is listed in Genbank under accession number U17253.

The sequences of the mouse and human NAB2 protein are described in Molecular and Cellular Biology, 1996 16, 3545-3553. The human DNA sequence is listed in Genbank under accession number U48361.

References to the NAB1 or NAB polypeptides and polynucleotides described hereafter are generally applicable to sequences of any origin, particularly the human sequences described above. As will be described below, the term NAB1 or NAB2 also includes variants, fragments and analogs of NAB1 or NAB2.

The following illustrative explanations are provided to facilitate the understanding of certain terms used here. The explanations are provided as convenience and are not limiting of the invention.

The biologically active fragments of NAB1 or NAB2 as referred to herein are those fragments that have an Egr-1 transcriptional repressor activity.

"GENETIC ELEMENT" generally means a polynucleotide comprising a region encoding a polypeptide or a polynucleotide region that regulates replication, rf-at- - ^ * ^ Bs ^? M? &?? &? -? rjf - -transcription or translation or other processes important for the expression of the polypeptide in a host cell, or a polynucleotide comprising both a region encoding a polypeptide and a region operably linked thereto that regulates expression. Genetic elements can be included within a vector that replicates as an episomal element, which is, like a molecule physically independent of the genome of the host cell. These can be included within the plasmids. Genetic elements can also be included within a genome of the host cell, not in its natural state, but, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector , among others.

HOSTED CELL is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.

IDENTITY, as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparison of the sequences. In the art, identity also means the degree of connected sequences between the polypeptide or polynucleotide sequences, as the case may be, determined by the coupling between the chains of such sequences. The identity can be really calculated (Computational Molecular Biology, Lesk, A: M., Ed., Oxford 5 University Press, New York, 1988, Biocomputing: Informa ti cs and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are a number of methods for measuring the identity between two polynucleotides or two polypeptide sequences, the term is well known to experts in the art. technique (Secjuence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H ., and Lipman, D., SIAM J. Applied Ma th., 48: 1073 (1988) .The methods commonly used to determine The identity between the sequences include, but are not limited to those described in Carillo, H., and Lipman, D., SIAM. J. Applied Ma th. , 48: 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the tested sequences. The ^ t «& ^^ ^ ^ ^ ^ ^ ^ ^ ^^^ rfU-áJT ^^ i ^ methods to determine identity are encoded in computer programs. Methods of the computer program for determining the identity between the two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASPT, BLASTN, and FASTA (Atschul, S: F., Et al., Molec. Biol. 215: 403 (1990)).

ISOLATED means altered "by the hand of man" from its natural state; that is, if it occurs in nature, it has been changed or eliminated from its original medium, or both. For example, a naturally occurring polynucleotide or a polypeptide that occurs naturally in a living organism in its natural state was not "isolated", but the same polynucleotide or polypeptide separated from the materials coexisting in its natural state was "isolated" , as the term is used here. As part of the following isolation, such polynucleotides can be linked to other polynucleotides, such as DNA, for example, by mutagenesis to form fusion proteins, and for propagation or expression in a host. Isolated polynucleotides, alone or linked to other polynucleotide sequences such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced within cells .. ^ «^ i ^ iOság? host in culture or in complete organisms, such DNAs that would be isolated, as the term was used herein, because they do not occur in their way or that are not compositions that occur in their natural environment, and polypeptides or polynucleotides remain there isolated within the meaning of the term as used here.

POLYUCLEOTIDE (S) generally refers to any polyribonucleotide or polideoxyribonucleotide, which could be unmodified RNA or DNA or modified RNA, DNA or cDNA. Thus, for example, the polynucleotides as used herein, refer inter alia, to single and double stranded DNA, DNA which is a mixture of single and double stranded regions or regions of a, double and triple strand, single- and double-stranded RNA, and RNA which is a mixture of single and double-stranded regions, hybrid molecules comprising DNA and RNA can be single-stranded, or more typically, double-stranded, or triple strand, or a mixture of single and double strand regions. In addition, the polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically include only one region of any of the molecules. One of the molecules of a triple helix region is often an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above, which contains one or more modified bases. Thus, spinal DNA or RNAs modified for stability or for other reasons are "polynucleotides" as the term is understood herein. Moreover, the DNAs or RNAs comprise unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, they are polynucleotides as the term is used herein. It should be appreciated that a wide variety of modifications have been made of DNA and RNA that serve any purpose known to those skilled in the art. The term "polynucleotide" as used herein encompasses chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA or RNA characteristic of viruses and cells, including simple and complex cells, inter alia. Polynucleotides encompass short polynucleotides often referred to as oligonucleotide (s).

POLYPEPTIDES, as used herein, include all polypeptides as described below. The basic structure of the polypeptides is well known and has been described in innumerable texts and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids linked together in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as polypeptides, oligopeptides and oligomers, and for example, to long chains, which are generally referred to in the art as proteins, of the which there are different types. It will be appreciated that polypeptides often contain different amino acids of the 20 amino acids commonly referred to the 20 naturally occurring amino acids, and that many amino acids, including terminal amino acids, can be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques that are well known in the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but are well described in texts basic and in more detail in monographs, as well as in extensive search literature, and one that is well known to those skilled in the art.

Among the known modifications that can be presented in the polypeptides for use in the present invention are, to name a few illustrative examples, acetylation, acylation, ADP-ribosylation, amidation, covalent binding of flavin, covalent attachment of a heme radical, covalent attachment of a nucleotide or nucleotide derivative , covalent binding of a lipid or lipid derivative, covalent binding of phosphotidylinositol, crosslinking, cyclization, formation of a disulfide bond, demethylation, formation of crosslinked covalent bonds, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic process, phosphorylation, prenylation, racemization, selenoylation, sulfation, mediated addition of RNA transfer amino acids to proteins such as arginilation, - and ubiquitination. Such modifications are well known to those skilled in the art and have been described in greater detail in the scientific literature. Several particularly common modifications, for example, glycosylation, lipid binding, sulfation, Gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation are described in many basic texts, such as, for example, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993). Various details Revised are available in this matter such as, for example, that provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifies tions and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). It will be appreciated, as is well known and as noted above, that the polypeptides are not always entirely linear. For example, polypeptides can generally be the result of post-translational events, which include a natural process event and events generated approximated by human manipulation that do not occur naturally. The circular, branched and circular branched polypeptides can be synthesized by natural processes of non-translation and also, by entirely synthetic methods. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common to occur naturally and synthetic polypeptides and also, such modifications * 5irt can be present in the polypeptides of the present invention. For example, the amino terminal residue of the polypeptides made in E. coli or other cells, prior to the proteolytic process, will be N-5 formylmethionine due to low invariance. During the post-translational modification of the peptide, a residue to the NH2-terminus can be removed. Accordingly, this invention contemplates the use of both the methionine-containing variant and the amino-terminal variants without methionine of the protein of the invention. The modifications that occur in a polypeptide will often be a function of how it is made. For polypeptides made by expression of a gene cloned in a host, for example, the nature and extent of the modifications will largely be determined by the The ability of post-translational modification of the host cell and the modification of signals present in the amino acid sequence of the polypeptide. For example, it is also known that glycosylation often does not occur in bacterial host cells such as, for example, example, E. coli Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylation host, generally a eukaryotic cell. The insect cells often carry out the same posttranslational glycosylation as the cells of mammal, and for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins that have native glycosylation patterns, inter alia. Similar considerations applied to other modifications. It will be appreciated that the same type of modification may occur in the same or different degree at several sites in a given polypeptide. Also, a given polypeptide may contain various types of modifications. In general, as used herein, the term "polypeptide" encompasses such modifications, particularly, those that occur in the polypeptides recombinantly synthesized by the expression of a polynucleotide in a host cell.

VARIANT (S) of polynucleotides or polynucleotides, as the term is used herein, are polynucleotides or polypeptides that differ from a polynucleotide or polypeptide reference, respectively. The variants in this sense are described below and thus also in the present description in greater detail. (1) A polynucleotide that differs in the nucleotide sequence from another reference polynucleotide. Generally, the differences are limited so that the nucleotide sequences of the reference and the variant are closely similar in their entirety, and in many identical regions, as noted below, the changes in the nucleotide sequence of the variant may be silent . That is, they can not alter the amino acids encoded by the polynucleotide. Where the alterations are limited to silent changes of this type, a variant will encode a polypeptide with the same amino acid sequence as the reference. Also as noted above, changes in the nucleotide sequence of the variant can alter the amino acid sequence of a polypeptide encoded by the polynucleotide reference. Such nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed above. (2) A polypeptide that differs in an amino acid sequence from another polypeptide reference. Generally, differences are limited because the sequences of the reference and the variant are closely similar in their entirety and, in many identical regions. A variant and a reference polypeptide may differ in an amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may occur in any combination.

The invention relates to the therapeutic uses of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a combination thereof. The invention also relates to therapeutic uses of fragments of said polynucleotide sequence encoding biologically active fragments of a NAB1 or NAB2 polypeptide, or variants of the polynucleotide sequence which, by virtue of its degeneracy of the genetic code, functionally encodes, that is, biologically active fragments of NAB1 or NAB2, and to functionally equivalent allelic variants and relates modified sequences by single or multiple base substitution, in addition and / or elimination encoding polypeptides having NAB1 or NAB2 activity.

These may be obtained by standard cloning procedures known to those skilled in the art.

Polynucleotides encoding transcriptional repressor proteins NAB1 or NAB2 may be in the form of DNA, cDNA or RNA such as mRNA obtained by cloning or produced by synthetic chemical techniques. The DNA can be one or two strands. The DNA of one strand can be the coding or sense strand (5 'direction) or it can be the non-coding or antisense strand (3' direction). For uses Therapeutics, the polynucleotide is in some way capable of being expressed to a functional NAB2 or NAB1 transcription repressor protein at the wound site in the subject to be treated. The polynucleotides can also be used by in vitro production of a NAB2 or NAB1 polypeptide for administration in a further therapeutic aspect of the invention as described in detail below.

The polynucleotides of the present invention that encode an NAB1 or NAB2 polypeptide may include but are not limited to the sequence encoding the NAB2 or NAB1 polypeptide, or biologically active fragments thereof. Thus, the polynucleotide can be provided together with additional non-coding sequences, including, for example, but not limited to non-coding sequences 51 and 3 ', such as the transcribed and non-transcribed sequences that play a role in transcription (including termination signals, for example), binding to the ribosome, mRNA stability elements, and additional coding sequences that encode additional amino acids, such as those that provide additional functionalities. The polynucleotides of the invention also include, but are not limited to, polynucleotides that comprise a structural gene for NAB1 or NAB2 and their associated genetic elements. naturally. Accordingly, the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a NAB1 or NAB2 polypeptide. The term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (e.g., disruption by integrated phage or insertion or editing sequence) together with additional regions, which may also contain coding sequences and / or do not code.

The present invention further relates to the variants of the polynucleotides described herein above which encode fragments, analogs and derivatives of the polypeptide. A variant of the polynucleotide can be a variant that occurs naturally such as a naturally occurring allelic variant, or it can be a variant that is not known to occur naturally. Such variants that occur unnaturally in the polynucleotide can be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.

Among the variants in relation to this are variants that IfoBfai = ñ-liSfcj ataMafcM »»!; ,.-¿¿Í > -r. üi-lg S & w- differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. Substitutions, deletions or additions may include one or more nucleotides. The variants can be altered in 5 codified or uncoded regions or both. Alterations in the regions they encode can produce conservative or non-conservative amino acid substitutions, deletions or additions.

Additional preferred embodiments of the invention are polynucleotides that are at least 70% identical in their entire length to a polynucleotide that encodes polypeptides having the amino acid sequence described hereinabove and the polynucleotides that are complementary to such polynucleotides. Alternatively, polynucleotides comprising a region which is at least 80% identical to its full length to a polynucleotide encoding a polypeptide of the present invention are more preferred. In relation To this, polynucleotides which are at least 90% identical in their entire length to it are particularly preferred. and among these particularly preferred polynucleotides, those which have at least 95% are preferred. Additionally, those with at least 97% are highly - «tfBBik« »i« M-riÍGG preferred among those with at least 95%, and among those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being most preferred.

Preferred embodiments in this regard, moreover, are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide NAB1 or NAB2 encoded by the DNA sequences described above (Genback access numbers U47007 and U48361) .

The present invention further relates to polynucleotides that hybridize to the sequences described hereinabove. In this regard, the present invention especially relates to polynucleotides that hybridize under severe conditions to the polynucleotides described above. As used herein, the term "stringent conditions" means a hybridization that occurs if at least 95% and preferably at least 97% identical between the sequences. Preferably the sequences that hybridize in this manner in the sequence of the invention encode a polypeptide having a biological activity of NAB1 or NAB2.

The polynucleotides can encode a polypeptide which is the additional amino plus of the mature protein or carboxy-terminal amino acids. Such additional sequences may play a role for example, they may lengthen or shorten the half-life or may facilitate the manipulation of a protein for a test or production, among other things. As is generally the case in vivo, the additional amino acids can be processed via the mature protein or by cellular enzymes.

Polynucleotides for use in gene therapy in the aspect of the invention, may be provided alone, or as part of a vector, such as an expression vector, examples of which are well known in the art.

A polynucleotide encoding NAB1 or NAB2 can be used therapeutically in the method of the invention via a therapeutic gene in which the polynucleotide is administered to a wound site or to another tissue that needs healing in a form in which it is capable of of directing the production of NAB1 or NAB2, or a biologically active fragment thereof, in situ. Preferably in genetic therapy, the polynucleotide is "ma &j?? á ií &? 3? a?" ^ Ái- ,. This method is administered such that it is expressed in the subject to be treated, for example, in the form of a recombinant DNA molecule comprising a polynucleotide encoding NAB1 or NAB2 operably linked to an acid sequence. nucleic that controls the expression such as in an expression vector. Thus, such a vector will include appropriate signals that include a promoter region capable of expressing the coding sequence, said promoter is operable in the subject to be treated. Thus, for human gene therapy, the promoter, which not only includes the term of the sequence necessary to direct the RNA polymerase to the transcriptional start site, but also, is appropriate, other operator or controller sequences that include improvements, preferably a human promoter sequence of a human gene, or of a gene which is typically expressed in humans, such as the human CMV promoter. Among the known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the late and early SV40 promoters, the retroviral LTR promoters, such as those of the Rous sarcoma virus ("RSV"). ), and metallothionein promoters, such as the mouse metallothionein-1 promoter.

A polynucleotide sequence and transcriptional control sequence can be provided cloned into a replicable plasmid vector, based on commercially available plasmids, such as pBR322, or can be constructed from available plasmids by the routine application of well-known published methods.

The vector may also include transcriptional control signals, located 3 'of the sequence encoding NAB2 or NAB1, and also in polyadenylation signals, of recognition in the subject to be treated, such as, for example, the corresponding sequences of viruses such as for human treatment, the SV40 virus. Other trans-regulatory control sequences that are well known in the art may be used. 5 Expression vectors may also include selectable markers, because of their antibiotic resistance, which allow the vectors to be propagated. 0 Expression vectors capable of synthesizing NAB2 or NAB1 if they can be introduced directly into the wound site by physical methods. Examples of these include the topical application of the "naked" nucleic acid vector in an appropriate vehicle, for example, in a solution in a pharmaceutically acceptable excipient, such as phosphate buffered saline (PBS), or administration of the vector by physical methods such as bombardment of particles, also known as "gene gun" technology, according to the methods known in the art, for example, as described in U.S. Patent No. 5371015 in which the inert particles, such as golden droplets covered with the vector are accelerated at a sufficient rate to allow and penetrate the surface 10 into the wound site , for example, skin cells, by means of discharge under high pressure of a projection device.

Other physical methods of administering the DNA directly to the container include ultrasound, electrical stimulation and electroporation.

A nucleic acid sequence encoding NAB1 or NAB2 for use in the therapy of the invention may also be administered by means of delivery vectors. These include viral delivery vectors, such as adenovirus or retrovirus delivery vectors known in the art.

Other non-viral delivery vectors include lipid delivery vectors, including liposome delivery vehicles, known in the art.

A nucleic acid sequence encoding NAB1 or NAB2 can also be administered at the site of the wound by means of transformed host cells. Such cells include cells harvested from the subject, wherein the nucleic acid sequence is introduced by gene transfer methods known in the art, followed by the growth of transformed cells in culture and grafting in the subject.

Expression constructs such as those described above can be used in a variety of forms in the therapy of the present invention. Thus, these can be administered directly at the subject's wound site, or they can be used to prepare a recombinant polypeptide NAB1 or NAB2 which can then be administered to the wound site as discussed in more detail below. The invention also relates to host cells that are engineered with constructs comprising NAB1 or NAB2 polynucleotides or polynucleotides of the present invention or genetic elements defined above, - * Vt * íí »- í¿¿? RS. . and to the uses of these vectors and cells in the therapeutic methods of the invention. These constructs may be used per se in the therapeutic methods of the invention or they may be used to prepare a NAB1 or NAB2 polypeptide for use in the therapeutic methods of the invention described in more detail below.

The vector can be for example, a plasmid vector, a single- or double-stranded phage vector, a single or double stranded DNA or RNA viral vector, which depends on whether the vector to be administered directly to the wound site (i.e. NAB1 or NAB2 in situ), or is to be used in the synthesis of recombinant NAB1 or NAB2. The primer plasmids described herein are either commercially available or publicly available, or they can be constructed from available plasmids by the routine application of well-known published methods. Many plasmids and other expression and cloning vectors that can be used in accordance with the present invention are well known and truly available to those skilled in the art.

Generally, vectors expressing a NAB1 or NAB2 polypeptide for use in the present invention comprise cis activation control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors are provided either by the host, provided by a complementary vector or provided by the same vector upon introduction into the host.

In certain embodiments in relation to this, the vectors provide a specific expression. For the production of NAB2 or recombinant NAB1, such specific expression can be expression or inducible expression only in certain cell types or both inducible specific cells. Particularly preferred among the inducible vectors are vectors that can be induced by expression by environmental factors that can be easily manipulated, such as temperature and nutrient additives. A variety of suitable vectors within the aspect of the invention include inducible expression vectors for use in prokaryotic and eukaryotic hosts, which are well known and are frequently employed by those skilled in the art.

A wide variety of expression vectors can be used to express NAB1 or NAB2 of the invention. Such vectors include, among others, chromosomal, episomal vectors, vectors derived from viruses, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion of elements, from chromosomal elements from yeasts, from viruses such as baculovirus, papova virus, such as SV40, vaccinia virus, adenovirus, skin damage virus in birds, seudorabies virus, and vectors derived from combinations of the The same, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, can all be used for expression according to this aspect of the present invention. Generally, any suitable vector to maintain, propagate or express Polynucleotides expressing a polypeptide in a host can be used for expression in relation to this.

The appropriate DNA sequence can be introduced into the vector by any of a variety of techniques such as, for example, those seen in Sambrook et al. , MOLECULAR CLONING, A LABORATORY MANUAL, 2ND Ed .; Cold Spring Hrbor Laboratory Press, Cold Spring Harbor, New York (1989).

The nucleic acid sequence in the expression vector is operably linked to the expression control sequence (s), which includes, for example, a promoter that directs the transcription of mRNA. Representative examples of such promoters include but are not limited to PL promoter from lambda phage 5, E. coli lac, trp and tac promoters, for recombinant expression, and the SV40 early and late promoters and promoters of retroviral LTRs for expression itself. you .

In general, the expression constructs will contain sites for the initiation of transcription and termination, and, in the transcribed region, a ribosome binding site for translation. The portion that encodes the mature transcripts expressed by the constructs will include a translation that starts AUG at the beginning and codon termination appropriately from position at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate as well as the expression of parenting. Generally, according to any of the commonly practiced procedures, such regions will operate by controlled transcription, such as transcription factors, sites that link repressors and termination, among others.

The propagation and expression vectors will generally include selectable markers and amplification regions, such as, for example, those below in Sambrook et al. , MOLECULAR CLONING, A 5 LABORATORY MANUAL, 2ND Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Representative representative samples of host suitable for the recombinant expression of NAB1 or NAB2 include cells bacterial, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cellus; fungal cells, such as yeast cells and Aspergill us cells; insect cells such as Drosophila S2 and cells Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.

The following vectors that are commercially available are provided by for example. Among the preferred vectors for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, vectors, Phagescript, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT40, pRIT5 available in Pharmacy, and pBR322 (ATCC ^^^^^^^ ^ ^ ^^^^^ 37017). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stragene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors can also be used for both a recombinant expression and an in situ expression that are listed only by the form of illustration in any of the commercially available manners and well-known vectors that are available to those skilled in the art to be used in accordance with this aspect of the present invention. It will be appreciated that any other suitable plasmids or vectors for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host cell can be used in this aspect of the invention.

Examples of vectors that are used in this aspect of the invention include expression vectors in which a NAB1 or NAB2 cDNA sequence is inserted into a plasmid, whereby the expression gene is driven from the immediate early cytomegalovirus enhancement promoter. of human (Foecking and Hosfstetter, Cell, 45, 101-105, 1986). Such expression plasmids may contain SV40 RNA that processes signals such as polyadenylation and termination signals. The expression constructs that use the CMV promoter and that ¿¿AiT ^ ^^ t ^^ fefeejjjriii ^ air ^^ E.rj. , i «r« -i ^ a ^. * ^. Aa & «are commercially available are pCDM8, pcDNA and derivatives, pcDNA3 and derivatives (Invitrogen). Other available expression vectors that can be used are pSVK3 and pSVL containing the SV40 promoter and site of mRNA site and SV40 polyadenylation signals (pSVK3) and SV40 VP1 processing signals (pSVL, Pharmacy vectors).

The promoter regions can be selected from any desired gene using vectors containing a reporter of unit transcript deficient to a promoter region, such as a transcription unit of chloramphenicol acetyl transferase ("CAT"), 3 'direction of restriction site or sites for the introduction of a candidate promoter fragment; that is, a fragment that can contain a promoter. As is well known, the introduction into the vector of a fragment containing a promoter at the site in the direction 51 of the production of spawning cat gene of the CAT, which can be detected by standard CAT tests. The appropriate vectors are well known and widely available, such as pKK232-8 and pCM7. Promoters for the expression of polynucleotides of the present invention include but are not only well known and indeed by available promoters, but also promoters that can be easily obtained by the prior art, using a reporter gene, for s-u expression in situ, such as a promoter should desirably be recognized in the subject to be treated.

Among the known prokaryotic promoters suitable for the expression of the polynucleotides and polypeptides according to the present invention are E. coli lacl lacZ and promoters, the T3 and T7 promoters. , the gpt promoter, the PR lambda promoter, the PL promoters and the trp promoter. Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly expressible gene to direct the transcription of a structural 3 'directional sequence, and a selectable marker to allow isolation of a vector containing cells after exposure to the vector.

The polynucleotides of the invention, which encode the heterologous structural sequence of a polypeptide of the invention will generally be inserted into the vector using standard techniques so that it is operably linked to the expression promoter. The polynucleotide will be positioned so that the transcription of the start site is properly located to a ribosome binding site. the ribosome tX &JsXm vii ?? Hi? í &? 0 ^^ 4Mkm ^ meí &? ? -. "* # *.? ¡¡¡¡¡¡¡¡" - - which links a site will be 5 'to the AUG, which initiates the translation of the polypeptide to be expressed.

Generally, there will be other open reading structures that start with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiation codon. Also, generally, there will be a stop codon of the translation at the end of the polypeptide and there will be a polyadenylation signal in constructs for use in eukaryotic hosts. The transcription of the appropriate termination signal is arranged at the end 31 at the end of the transcribed region that can also be included in the polynucleotide construct.

For the secretion of the translated protein in the lumen of the endoplasmic reticulum, or in the periplasmic space or within its extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide when they are recombinantly synthesized. These signals may be endogenous to the polypeptide or these may be heterologous signals.

The polypeptide can be expressed in a modified form, such as the fusion of a protein, and can be included not only in the secretion of the signals but also in the additional heterologous functional regions. Thus, for example, an additional amino acid region, particularly charged , can be added to the N- or C-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or duration, subsequent handling and storage. Also, the region can be added to the polypeptide to facilitate purification. Such regions can be eliminated prior to the end of the polypeptide preparation. The addition of peptide radicals to the polypeptides to generate secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous immunoglobulin region that is useful for solubilizing or purifying the polypeptides. The cells are typically harvested by centrifugation, broken down by physical and chemical means, and the resulting crude extract is retained by further purification.

The microbial cells in the expression proteins can be broken by any of the convenient methods, including freezing cyclization, sonication, mechanical disruption, or the use of cell lysis agents, Fe ^ s * jyH | sMsí * ¡j ^^ such methods are well known to those skilled in the art. Mammalian expression vectors may comprise an origin of replication, a suitable and improved promoter, and also necessarily any ribosome binding site, polyadenylation regions, acceptor and splice donor sites, transcriptional termination sequences, and flanking non-transcriptional sequences to 5 'that are necessary for the expression.

To prepare NAB1 or NAB2 polypeptides for use in the invention of genetic engineering, host cells can be used. It can affect the introduction of a polynucleotide into the host cell by transfection of calcium phosphate, DEAE-dextran mediated transfection, transfection, microinjection, lipid-mediated cationic transfection, electroporation, transduction, scraping charge, ballistic introduction, infection or other methods. Such methods are described in any of the standard laboratory manuals, such as Davis et al., BASIS METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Mature proteins can be expressed in host cells that include mammalian cells such as CHO cells, yeast, bacteria, or other cells under the control of suitable promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Expression and cloning vectors suitable for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The polypeptide can be recovered and purified from the cell culture by well-known methods including precipitation of ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. more preferably, high performance liquid chromatography is used for purification. Well-known techniques for the refolding protein can be employed to regenerate the active conformation when the polypeptide is denatured during isolation and purification.

S * a »r» .j, t "^ ne ^ sA ^ .i Therapeutically, a polynucleotide encoding NAB1 or NAB2, for example in the form of a recombinant vector, can be purified by techniques known in the art, by such as column chromatography as described in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL 2ND Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

As indicated above, an NAB1 or NAB2 polypeptide can be administered at the site of its wound as in the nucleic acid encoding NAB2 or NAB1 which is transcribed and translated into NAB2 or NAB1 at the same wound site in a form of therapy of the gene, or the transcriptional repressor protein thereof can be administered directly.

Thus, according to a further aspect of the invention, there is provided the use of a NAB2 or NAB1 polypeptide, or a biologically active fragment thereof, in the manufacture of a medicament for the treatment of proliferative cell diseases in a mammal , which includes the human.

According to a further aspect of the present invention, .t ... ¿ltJfett, ¡,.

A method of treating proliferative cell disorders in a mammal, including a human, which comprises administering to the mammal a therapeutically effective amount of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, is provided.

Thus, viewed from a further aspect, the invention provides the use of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for use in the treatment of wounds and wound healing.

As used herein, the term "NAB2 or NAB1 polypeptide" includes analogue or biologically active, natural and synthetic variants of NAB1 or NAB2 produced recombinantly and naturally or derivatives thereof or biologically active fragments and variants, derivatives and analogs thereof fragments The products of the NAB1 or NAB2 protein include biologically active fragments of NAB1 or NAB2, which can be generated and / or isolated generally by techniques known in the art.

The NAB1 or NAB2 and the aforementioned fragments and derivatives of the same for use in the therapy of the invention can be extracted from natural sources by methods known in the art. Such methods include purification by means of affinity-specific sequence chromatography of DNA using methods such as those described in Briggs et al, Science 4 234, 47-52, 1986, using DNA that binds oligonucleotides with recognized NAB1 or NAB2. The polypeptide can also be prepared by methods of technology known in the recombinant DNA art as described above, that is, by expression in host cells of the described constructs. Alternatively, the polypeptides of the invention can be produced synthetically by conventional peptide synthesizers.

The invention also relates to the use of analog fragments and derivatives of NAB1 or NAB2. The terms "fragment", "derivative" and "analogue" mean a polypeptide that retains essentially the same function or biological activity of such a polypeptide. Thus, an analog includes a proprotein that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The fragment, derivative or analogue of the polypeptide can be £ ¿.%. (i) one in which one or more of the amino acid residues is substituted with a conservative or non-conserved amino acid residue (preferably a conserved amino acid residue) and such conserved amino acid residue may or may not be encoded by the gene encoder, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, the Glycol polypeptide), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or a secretory or guiding sequence or a sequence in which it is used for the purification of the mature polypeptide or a proprotein sequence, such fragments, derivatives and analogs are considered within the scope of those skilled in the art from the aforementioned teachings.

Among the preferred variants are those that naturally occur as NAB1 or NAB2 by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide with another amino acid of similar characteristics. Typically seen as conservative substitutions that are replaced one by another, between the aliphatic amino acids Ala, Val, Leu and lie; exchange of the hydroxyl residues Ser and Thr, exchange of the acid residues Asp and Glu, the substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replaced between the aromatic residues Phe, Tyr.

Additionally, variants, analogs, derivatives and fragments, and variants, analogs and derivatives of fragments, having the amino acid sequence of the polypeptide in which several, a few, from 5 to 10, of 1 to 5, from 1 to 3, 2.1 or no amino acid residue are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptide of the present invention. Also, conservative substitutions are especially preferred in this regard.

Fragments which are biologically active fragments ie fragments which retain the wound healing properties of the matrix polypeptide are particularly preferred.

- - The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified by homogeneity.

The NAB1 or NAB2 polypeptides of use in the present invention include NAB1 or NAB2 polypeptides as well as polypeptides having at least 70% identity, preferably at least 80% identity and even more preferably at least 90% identity identity and yet still more preferably at least 95% similarity (even more preferably at least 95% identity) in the polypeptide sequences described above and also include portions of such polypeptides with a portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention can be employed for the corresponding production of full-length polypeptide by peptide synthesis; therefore, the fragments can be used as intermediates for the production of the full-length polypeptides. The fragments or portions of the polynucleotides of the present invention can be used to synthesize the full length polynucleotides of the present invention.

The invention also relates to the use of NAB1 or NAB2 polypeptide fragments defined above and fragments of variants and derivatives thereof.

In this regard a fragment is a polypeptide having an amino acid sequence that in its entirety has the same part but not the entire amino acid sequence of polypeptide NAB1 or NAB2 and variants or derivatives thereof.

Such fragments can be "dye-free", ie, they do not split or fuse from other amino acids or polypeptides, or they can be included within a long polypeptide or that can form a part or region. When included within a long polypeptide, the fragments discussed more preferably present a single continuous region. However, several segments can be included within a longer polypeptide. For example, certain preferred embodiments refer to a fragment of a polypeptide of the present invention included within a polypeptide designated for expression in a host and having heterologous pre- and pro-polypeptide regions fused to the amino terminus or to the fragment and additional au-ionion fused to the carbomyl terminus of the fragment. Therefore, the fragments in one aspect of the meaning intended herein, refers to the portion or portions of a polypeptide or fusion protein fusion derived from a polypeptide of the present invention.

Fragments characterized by their functional and structural attributes of the polypeptide of the present invention are also preferred in this aspect of the invention. Preferred embodiments of the invention in relation to this include fragments comprising alpha helix and alpha helix forming regions, beta sheets and beta sheet forming regions, spirals and spiraling regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface forming regions, regions that bind substrates, and high antigen index regions of the polypeptide of the present invention, and combinations of such fragments.

Preferred regions are those that mediate the activities of the polypeptide of the present invention. HE .--., - ^ - ^ - 1 & ^ -: ^ j & amp; amp; amp; & amp; amp; amp; & amp; amp; amp; & amp; amp; amp; & amp; amp; amp; amp; & amp; amp; & amp; amp; amp; amp; amp; amp; similar activity or an improved activity or with a decrease in unwanted activity. Additionally, preferred polypeptide fragments are those that comprise or contain antigenic or immunogenic determinants in an animal, especially in a human.

It will be appreciated that the invention also relates to, inter alia, polynucleotides encoding the above-mentioned fragments, polynucleotides that hybridize the polynucleotides encoding the fragments, particularly those that hybridize under severe conditions, and polynucleotides, such as PCR primers, to amplify polynucleotides. that encode the fragments. In this regard, polynucleotides are preferred which are those corresponding to the preferred fragments, as discussed above.

In further embodiments of this aspect of the invention, they include biologically, prophylactically, clinically or therapeutically useful, analogous or derivative variants thereof, or fragments thereof or variants iA * «4 * *? tteii &amfi! MHiSj8S & + ~ > '' "'.-1A> • i ^ ^ m ^^ J ^^ K-í ^^^ - ^ -xí-ínsS' analogous and derivatives and compositions comprising the same.The biologically active, analogous variants or fragments they are included within the scope of the present invention.

The invention also relates to compositions comprising the polynucleotides or polypeptides of the present invention that can be used in combination with a pharmaceutically acceptable carrier or carriers.

Such carriers may include, but are not limited to, saline, saline buffer, dextrose, water, glycerol, ethanol, and combinations thereof.

The polypeptides and polynucleotides can be employed in the present invention, either alone or in conjunction with other compounds such as therapeutic compounds.

The pharmaceutical compositions can be administered in any effective and convenient manner to form wound sites including, for example, routes of topical, intravenous, intramuscular, intranasal or intradermal administration among others. In therapy or as a prophylactic, the active agent is > < to? »Ja? ? *? a? JM - - can be administered to an individual with an injectable composition, for example as a sterile, preferably isotonic, aqueous dispersion.

Alternatively, the composition can be formulated by topical application, for example in the form of ointments, creams, lotions, eye ointments, eye drops, drops, rinses, impregnated dressings and sutures and aerosols, and may contain additives conventional ones, including, for example, condoms, solvents that help penetrate the drug, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example creams or bases, and ethanol or alcohol for oily lotions. Such carriers can constitute from about 1% to about 98% by weight of the formulation; but usually they will constitute up to approximately 80% by weight of their formulation.

For administration in mammals, and particularly in humans, the level of the daily dose of the active agent is expected to be from 0.01 mg / kg to 10 mg / kg, typically between lmg / kg. The doctor in any event will determine the current dose that will be most appropriate individually and will vary with the age, the weight and response of the 'léj rticular. The doses above are exemplified by the average number of cases. It may of course be in cases where the weight or low dose ranges are necessary, and are within the scope of the present invention. Finally, the ítiosis selected by those skilled in the art will be the function of reducing cell proliferation without preventing wound healing.

As a further aspect, there is provided a pharmaceutical composition comprising a NAB1 or NAB2 polypeptide or a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, together with one or more pharmaceutically acceptable carriers thereof.

The therapeutic advantage of using transcriptional repressor proteins in wound healing is the deactivation of multiple target genes that promote accelerated healing. NAB1 or NAB2 is naturally activated in response to healing and is also an advantage of the increase in response. The treatment is based on DNA and provides a reliable and reproducible delivery system.

When a NAB1 or NAB2 polynucleotide is used in the therapeutic method of the invention, the polynucleotides can be used as part of a construct expression for example, in the form of an expression vector. In a method such that the construct is introduced to the wound site where NAB1 or NAB2 is produced in situ. The constructs used can be standvectors and / or gene delivery systems, such as liposomes, mediated receptor delivery systems and viral vectors.

The present invention is suitable for all aspects of wound healing including limb ulcerations, in diabetes and peripheral arterial occlusive conditions, psoriasis, inhibition of restenosis followed by percutaneous trans-luminal coronary angioplasty, modulation of wall calcification of the vessels and inhibition of cell proliferation in cancer.

As described above, the NAB1 or NAB2 polypeptides or nucleic acids of the present invention are administered locally at the site of the damaged tissue by any convenient method for example, by topical administration. A preferred delivery method of delivery of nucleic acid products is used in the gene gun technology in which the "isolated" Egr-1 nucleic acid molecule for example, in the form of cDNA or in an expression vector is ' ^^ aS ^ ^ fi ^ i ^^ it ^^ iíÉ ^ .it ^ tr¿ ^ k ^ - > . % ^ gsk ^ í ** ^^ j ^ ^^^^ l ¡8 ^^^^^^^ & l ^ ^ ^ * immobilizes in gold particles and arrives directly at the site of the wound. Thus, as a preferred aspect of the present invention, there is provided the use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide in a gun gene for the treatment of cell proliferative conditions associated with the healing of the wound Additionally, a composition suitable for gene therapy comprising a sequence encoding NAB1 or NAB2 immobilized on gold particles is provided.

The present invention will be described by way of the following unlimited examples with reference to the accompanying figures, wherein: The figures la) - Id). shows the repression of the Egr-1 mediated activation of PDGF-AB, TGFβ, HGF and VEGF respectively; Figure 2 depicts the trans-repression of NAB2 from the production of HGF mediated by Egr-1 in HVSMC; Figure 3 describes the effect of NAB2 on angiogenesis driven by Egr-1; Figure 4a describes the effect of NAB2 transfection on a linear incisional wound contraction at 7 days after healing; Figure 4b describes the effect of transfection of NAB2 on the levels of growth factors in the epidermis of 7-day rat incisional wounds.

Figure 4c describes the effect of transfection of NAB2 on the levels of growth factors in the granulation of the tissue of incisional wounds in rat 7 days; Y Figure 4d describes the effect of NAB2 transfection on angiogenesis in 7-day rat incision wounds. EXAMPLES Example 1 Use of NAB2 to repress Egr-1 mediated trans-activation of growth factors. 20 1.1 Methods Human vascular smooth muscle cells (HVSMC; Cloning) are cultured according to the recommendations of the manufacturers. The cells are cultured by transfection in 6-well titration plates (Costar). An expression plasmid comprising the NAB2 c DNA driven by the human cytomegalovirus promoter (hCMV, Svaren et al Mol Cell, Biol., 16; 3545-3553) was transfected together with a expression plasmid comprising cDNA Egr-1 (Houston et al Arterioscler, Thromb. Vasc. Biol., 19; 281-289) in HVSMC in the relationship described in the figures using FUGENE (Boehringer Mannheim). It was used in a ratio of 3: 1 (volume / weight) of FUGENE: DNA for all experiments and the The transfection was normalized using a β-galactoid-β-CMV expression plasmid. The secreted growth factors are detected in a tissue culture medium by ELISA (R & D Systems) using appropriate controls. 1.2 Results Figures la) to Id). show the repression of Egr-1 mediated by the activation of PDGF-AB, TGFβ, HGF and VEGF respectively. 6 μg of Egr-1 expression plasmid was affected either by (figure, figure lb and figure Id) or by 500, 100 or 25 ng of NAB2 (figure and figure Id). For PDGF-AB, TGFβ and VEGF, little was increased in the amount of secreted growth factor detected when Egr-1 was transfected alone. In the co-transfection with NAB2, it was completed the ablation of the PDGF-AB response for the activation of Egr-1, for the activation and reduction of expression? Basal expression that leads to a 5-fold decrease in the total production of PDGF-AB (figure). The transfection of 5 NAB2 completely lowered the TGFβ response to the activation of Egr-1 and caused a 30% reduction in the total production of TGFβ (Figure lb). Using the DNA concentrations shown in the figure, the transfection of Egr-1 caused the 50% increase in HGF production which was blocked partially by co-transfection of a low dose of NAB2 and completely blocked by a high dose (Figure lc). The increase in effects is seen on the first day after transfection, at the same time as it inhibits the effects of NAB2 that are apparent at 2 and 3 days. The transfection of NAB2, as with PDGF-AB and TGFβ blocked by Egr-1 mediated activation of VEGF and that caused a 40% decrease in the total amount of VEGF produced (Figure Id). 1.3 Conclusion These data show that the Egr-1 suppressor of NAB2 can block the activation of the growth factor mediated by Egr-1, as it is found in sites of acute damage.

^^^^^^^^^^^^^^^^ ^^^^^^^^ Example 2 Use of NAB2 to suppress the basal levels of growth factors 2. 1 Methods Cell culture, transfection and detection of growth factors are carried out as described in section 1.1 above. The NAB2 expression vector was transfected at the end of the concentrations of 0, 250, 500, 1000 or 3000 ng. 2. 2 Results As shown in Figure 2a, the transfection of NAB2 within the HVSMC causes a drastic reduction in the production of PDGF-AB. A high dose of NAB2, a 10-fold reduction in PDGF-AB was obtained. 2. 3 Conclusion These data show that the Egr-1 suppressor can reduce the basal levels of growth factors produced by both PDGF-AB promoters.

Example 3 Use of NAB to suppress induction of angiogenesis in vi tro 3. 1 Methods The expression constructs of activated NAB2 (dominant wild type 5) of the hCMV promoter have been described (Svaren et al, EMBO J. 17; 6010-6019, 1998). It has been demonstrated that transfection of the transcription factor Egr-1 when expressed by angiogenesis promoted by the hCMV promoter (Patent filing PG3412). In these experiments The DNA was transfected into an angiogenesis kit as provided and maintained according to the manufacturer's instructions (TCS Biologicals). The VEGF protein (2 ng / mL) was used as a positive control and suramin (20 μM) as an inhibitor of angiogenesis. The CMV Egr-1 DNA is transfection at 0.5 μg per well in triplicate in a 24-well microtiter plate using a Transit Mirus reagent (Cambridge Biosciences) in a ratio of 2: 1 volume / weight of DNA. To test the potential of NAB2 repressor of Egr-1 mediated with angiogenesis, 0.5 mg of Egr-1 DNA was co-transfected with 10, 25 or 100 ng per well, the plasmid of expression of NAB2 in the absence or presence of lOOng of a dominant negative expression plasmid. After 11 days of the co-culture of the angiogenesis, cell staining was determined for the endothelial cell marker PECAM-1 and the ^^ - ^ r., -, ~ ^ .t -r 'irr display using the BCIP / NBT substrate.

Representative images of tubule formation using four doses of Egr-1 expression plasmid together with VEGF (positive control) and suramin (negative control) were captured and processed by image analysis using Quantimet 600 image analyzer and associated programs. 3. 2 Results 10 It has been shown that the DNA of Egr-1 is pro-angiogenic in the International Patent Application Number PCT. GB99.01722 (as shown in column 4 of figure 3a). As shown in Figure 3a, co-transfection with NAB2 gave a dose that depends on the reduction in the ability of Egr-1 to induce angiogenesis, which was partially eliminated by co-transfection with the dominant negative NAB23. 3 Conclusion NAB2 can be used to block angiogenesis in the antecedent of acute damage, an example of which is the induction of growth factor by Egr-1.

Example 4 Use of NAB2 to reduce healing in a model incisional healing of a rodent, 4. 1 Methods 4. 1.1 The effect of transfection of NAB2 using the gene gun on growth factor levels of incisional wounds in rat The transfection of NAB2 within the rodent incision wounds has the potential to be anti-staining through its suppressive action on the expression of the staining key of growth factors, especially TGFßl. In this experiment, the NAB2 cDNA was transfected into rat blunt wounds using the Biorad gene gun and the effects on healing of growth factor levels were imposed using routine histology and immunocytochemistry. 4. 1.2 Mediator gene transfer by particles Eight male Sprague Dawley rats weighing 250 g were anesthetized under isoflorane in a 2: 1 ratio of oxygen / nitrogen oxide. Two transfection sites (5 cm from the base of the skull, 1.5 cm from the side of the midline) and 2 control sites (8 cm from the base of the skull 1.5 cm either from the side of the midline) on the back of the rat first prepared the skin trimming, then shaved with a shaver. A transfection was carried out at each transfection site by accelerating plasmid / gold complexes of either NAB2 or Egr-1 (positive control for growth factor activation) within the skin at 350 psi. Typically 0.5 -1.5 μg of DNA was delivered by transfection. At a control site, gold particles (without DNA) are accelerated into the skin at 350 psi; the other control site was left untreated. The transfection sites and control sites were rotated clockwise within each additional animal to control each previous and subsequent difference in the healing of the wounds of each rodent. 4. 1.3 Healing Model in Incision Wounds Twenty-four hours after transfection, the animals were anesthetized and a full linear 1 cm thick incision was made parallel to the spine, using a scalpel blade at the exact transfection sites. The animals were allowed to recover from the anesthesia and the wound was allowed to heal free of sutures. At 7 days after wounding all 8 animals, they were killed and the wounds bisected and harvested by routine histology and immunohistochemistry. 4. 1.4 Histological Analysis Each fresh wound after dissection was bisected 5 horizontally. One half was placed in 4% paraformaldehyde for 24 hours and processed for wax histology. Sections of 5 mm of each wound were cut using a microtome and sections were stained with van Geison. The sections were observed using light microscopy and the effect of NAB2 on the healing response evaluated. 4. 1.5 Immunohistochemistry Once frozen in OCT, the second half of each wound was sectioned at 7 mM using a cryostat. Two sections of each wound were fixed in ice-cold acetone and fluorescent immunoblotting performed with a primary antibody of Egr-1, PDGF, TGFβ1, TGFβ3 and WFv using the following protocol. 1. Wash the sections with PBS 2. Apply 30 mL of primary antibodies for 1 hour. 3. Wash 3 x 5 minutes in PBS 4. Apply 30 mL of secondary antibody (directly linked FITC) for 45 minutes.

. Wash 3 x 5v minutes in PBS 6. Cortes mounted using aqueous mount Immediately after immunoblotting each cut was placed under a fluorescence microscope and the wound area was captured using an amplification x 100. The image was integrated and the threshold adjusted to minimize the background. The area and intensity of the spotting was measured using an image analysis and a graph was formed. 10 4.2 Results 4. 2.1 Effect of NAB2 on the healing of incisional rat wounds 15 4.2.1.1 Wound contraction and Histological analysis of 7-day wounds According to Figure 4a, the wound treated with NAB2 contracted to a similar width as that transfected by Egr. - 1 and the control wounds, showed histologically similar granulation in the mature tissue.

Conclusion The supply of NAB2 did not impair the speed of healing in a rodent incision model. ^^^^^^^^^^^^^^^^^^^^^^ m ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^ 4.2.1.2 Growth factor profile The immunoblot for Egr-1, TGFßl, TGFß3 and PDGF-AB performed in frozen cryosections of the skin tissue 5 at the wound site and the intensity of spotting for each growth factor was measured using image analysis. Two measurements were made separately within the wound site. The sections were examined for the expression of the growth factor in both, both in the epidermis 10 immediately before the wound and within the wound site (granulation tissue). a) Positive spotting for growth factors in the epidermis 15 As shown in Figure 4b, at 7 days after the NAB2 cure, the expression of TGFßl in the epidermis compared to Egr-1 and only the control is markedly reduced of gold. In comparison to the unmanipulated control, the transfection NAB2 reduced the expression of TGFβ1 in the epidermis, while in both, the Egr-1 and the gold supply activated the production of TGF ßl. The transfection NAB2 increased the levels of TGF ß3 in the epidermis compared to both, gold alone and unmanipulated controls. The transfection of NAB2 did not alter the expression markedly epidermal of Egr-1 and PDGF. b) Positive staining of the growth factors in the granulation tissue As shown in Figure 4c, at 7 days after the wounds the transfection of NAB2 had no effect on the levels of Egr-1, PDGF-AB and TGFβ within the granulation of the tissue. However, NAB2 increased levels of TGF ß3 within the granulation tissue compared to both gold alone and unmanipulated controls.

Conclusion Transfection of NAB2 in incisional wounds decreases the levels of TGFbl in the epidermis and increases the levels of TGF-β3 in the epidermis and granulation tissue at 7 days after wounding. TGF ßl is a known healing, while TGF ß3 has anti-chalking properties (Shah et al J. Cell Science 107; 1137-1157, 1994). Therefore, the NAB2 supply may have anti-healing properties. 4. 2. Effect of NAB2 on angiogenesis At 7 days after wounding, sections of the skin ^^ S are stained and a record is used for the vWF expression using immunohistochemistry and image analysis. Angiogenesis is quantified using a von Willebrand immunoblotting factor in wound cryoses and image analysis to measure the positive staining area within the wound site. As shown in Figure 4d, 7 days after the wound, the transfected NAB2 wounds had fewer new blood vessels compared to the control (wounds treated with gold). Egr-1 promoted angiogenesis in vivo, supporting the in vitro findings in Example 3.

Conclusion NAB2 blocked the stimulated activation of Egr-1 angiogenesis in vivo, supporting a role of NAB2 as a repressor of growth factor activation when activated by an acute stimulus, an example of which is the production of growth factor of activation of Egr-1.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property: : ^ t ^? Xíi, rSüte-T-KSt -. ' r. aí¿? .jt S = fe ^^ fe

Claims (22)

1. CLAIMS 1. The use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a medicament for the treatment of proliferative diseases of cells associated with the healing of a wound in a mammal, which includes the human.
2. 2. The use as claimed in claim 1, characterized in that the NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide.
3. 3. The use as claimed in claim 1-2, characterized in that the proliferative cell diseases associated with wound healing are hypertrophic and keloid formations.
4. 4. The use as claimed in any of claims 1-3, characterized in that the nucleic acid molecule is operably linked to a nucleic acid sequence, which controls expression.
5. 5. The use as claimed in any of claims 1-4, characterized in that the molecule of the The nucleic acid is at least 70% or 80% or 90% or 95% identical to its total length in a polynucleotide sequence of NAB1 or NAB2.
6. 6. The use according to any of claims 1-5, characterized in that it comprises sequences encoding both, a NAB1 polypeptide and an NAB2 polypeptide, or biologically active fragments thereof.
7. 7. The use as claimed in claims 1-5, characterized in that the nucleic acid molecule comprises a sequence encoding a NAB2 polypeptide, or a biologically active fragment thereof.
8. 8. The use as claimed in claims 1-7, characterized in that the nucleic acid molecule is stopped for administration to the mammal by physical methods.
9. 9. The use as claimed in claim 8, characterized in that the nucleic acid molecule is administered to the mammal by bombardment of the particle.
10. 10. The use as claimed in claim 9, characterized in that the nucleic acid molecule is immobilized in gold particles.
11. 11. The use as claimed in claim 8, characterized in that the nucleic acid molecule is arranged for administration by microsembration.
12. 12 The use as claimed in claim 1-7, characterized in that the nucleic acid molecule is a vector.
13. 13. The use as claimed in any of claims 1-7, characterized in that the nucleic acid molecule is in a cell.
14. 14. A nucleic acid molecule characterized in that it comprises a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for use in gene therapy.
15. 15. A pharmaceutical composition characterized in that it comprises a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, together with one or more pharmaceutically acceptable carriers thereof.
16. 16. A method of treating the conditions of cell proliferation associated with a wound healing of a mammal, including the human, characterized in that the method comprises administering to the mammal a nucleic acid molecule comprising a sequence encoding a polypeptide NAB1 or NAB2, or a biologically active fragment thereof.
17. 17. The use of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a medicament for the treatment of cell proliferation conditions associated with wound healing in a mammal, including the human.
18. 18. The use as claimed in claim 17, characterized in that the NAB1 or NAB2 polypeptide or biologically active fragment thereof is produced naturally, synthetically or recombinantly.
19. 19. The use as claimed in claim 17 or claim 18, characterized in that the NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide.
20. 20. The use as claimed in claims 17-19, characterized in that the polypeptide is at least 70% or 80% or 90% or 95% identical in its total length to a polynucleotide sequence NAB1 or NAB2.
21. 21. A method for the treatment of the diseases of cell proliferation with wound healing in a mammal, including human, characterized in that it comprises the administration to the mammal of a therapeutically effective amount of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof. .
22. 22. A pharmaceutical composition characterized in that it comprises a NAB1 and / or NAB2 polypeptide, or a biologically active fragment thereof, together with one or more pharmaceutically acceptable carriers thereof.